Embedding muscle fibers in hydrogel improves viability and preserves contractile function during prolonged <i>ex vivo</i> culture

Ex vivo culture of isolated muscle fibers can serve as an important model for in vitro research on mature skeletal muscle fibers. Nevertheless, this model has limitations for long-term studies due to structural loss and dedifferentiation following prolonged culture periods. This study aimed to investigate how ex vivo culture affects muscle fiber contraction and to improve the culture system to preserve muscle fiber morphology and sarcomere function. Additionally, we sought to determine which culture-induced changes can negatively affect muscle fiber contraction. We cultured isolated flexor digitorum brevis (FDB) muscle fibers in several conditions for up to 7 days and investigated viability, morphology, and the unloaded sarcomere shortening in intact fibers, along with force generation in permeabilized muscle fibers. In addition, we examined changes to the microtubule network. We found a time-dependent decrease in contractility and viability in muscle fibers cultured for 7 days on a laminin-coated culture dish (2D). Conversely, we found that culturing FDB muscle fibers in a low-serum, fibrin/Geltrex hydrogel (3D) reduces markers of muscle fiber dedifferentiation (i.e., sprouting), improves viability, and retains contractility over time. We discovered that the loss of contractility of cultured muscle fibers was not the direct result of reduced sarcomere function but may be related to changes in the microtubule network. Collectively, our findings highlight the importance of providing muscle fibers with a 3D environment during ex vivo culture, particularly when testing pharmacological or genetic interventions to study viability or contractile function.